The project described was supported by Grant Number R01HD050725 from the National Institute of Child Health and Human Development to T.A.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Child Health and Human Development or the National Institutes of Health. We thank members of the Godenschwege lab as well as Barbara Schreader for their input to the manuscript and video.

1. Dissection of the ventral nerve cord
Dissection and dye filling of the GF can be performed at room temperature. For all dissection steps cold (4&deg;C) saline should be used to slow down cell metabolism and keep the neurons alive longer.
<ol>
 <li>Anesthetize adult flies (4 days or older) with CO2 or chill them on ice until they are immobilized. Anesthetizing with ice will require more 
 time (up to 30 min) but will keep the flies immobilized for longer.</li>
 <li>Place one fly in a Sylgard coated Petri dish. This dissection requires the use of a high magnification (80-100X) dissection stereomicroscope.</li>
 <li>With Vannas scissors, remove legs, proboscis, and wings.</li>
 <li>Use coarse forceps and fine insect pins (minutien pins) to orient the fly dorsal side up in the Sylgard dish. Pin the abdomen first and then elongate the animal 
 slightly by placing two pins on each side of the neck, behind the head, so that the neck is slightly stretched and accessible (see Figure 2).</li>
 <li>Submerge the fly in fresh, cold Drosophila saline. Most commonly used Drosophila salines are sufficient for the dissection, however here we used a 
 saline described in Gu and O'Dowd, 20064. An air bubble will surround the fly. Remove the air with a syringe.</li>
 <li>Make a short, lateral incision into the dorsal end of the abdomen. Starting at the incision, make a shallow, straight cut along the dorsal midline of the 
 abdomen towards the thorax. Then continue the shallow, longitudinal cut in the thorax as precisely as possible along the midline toward the connective. It 
 is critical to make the cuts shallow as to not damage the VNC (Figure 1).</li>
 <li>Use two fine insect pins to carefully open the thorax. Spread the body halves apart using the pins and place the pins through the flight muscles (Figure 2). 
 The thorax should not be opened further as depicted in the Figure 2 to avoid ripping of the cuticle. To ensure a straight orientation of the VNC the pins 
 should be placed in the same anterior posterior position on each side.</li>
 <li>Access to the VNC for dye injection and proper imaging may be obstructed by internal organs. Therefore, use forceps and scissors to remove the stomach, gut, 
 heart, and reproductive organs. Pull out the salivary glands lying on both sides of the VNC. Rinse the dissection carefully with fresh saline to remove 
 floating tissue and fat.</li>
 <li>Remove the fat body from underneath the cervical connective (CvC). The fat body is best visible at a high magnification. Use fine forceps to carefully 
 grasp the tissue from the side and pull it out.</li>
</ol>
The dissection can be performed within 5 to 10 minutes. By using fresh, cold saline the dissected, undamaged nervous system will remain alive for at least one hour.
2. Intracellular GF fill
It is recommended to perform the dye filling as soon as possible after the dissection.
<ol>
 <li>Place the Sylgard dish with the dissected fly under an upright microscope with a fixed stage (Figure 3) and a 10x air objective. Center the sample 
 with the posterior end of the fly positioned toward the dye fill electrode holder. Use the differential interference contrast (DIC) mode and focus on the 
 CvC. The big axons of the GFs should be clearly visible on the dorsal surface of the CvC.</li>
 <li>Dilute Lucifer Yellow CH dilithium salt in deionized H2O to a concentration of 1%. Pull a Borosilicate glass electrode with filament to a resistance 
 of approx. 60-80 M&Omega;. The tip of the glass electrode should be long and sharp. Fill the tip of the electrode with 1% Lucifer Yellow solution by placing 
 the electrode with the end opposite the electrode tip into the Lucifer Yellow solution for approx. 30-60 seconds. Then backfill the electrode with 3M LiCl 
 using a Hamilton syringe leaving an air bubble between the dye and LiCl. Connect the dye fill electrode to the head stage.</li>
 <li>Insert a chloride coated silver wire ground electrode into the saline and place the dye fill electrode above the fly parallel to the VNC using a 3D hydraulic micromanipulator. The dye fill electrode should be connected to an amplifier that allows passing current in order to release negatively or positively charged dyes from the electrode by passing hyper- or depolarizing current, respectively.</li>
 <li>Switch to a 40x water immersion lens and lower the tip of the dye fill electrode onto one of the GFs. The GF axons lay close to the dorsal surface of the CvC. Use a forward (toward the head) movement to insert the electrode into the GF.</li>
 <li>Change your light source to epifluorescent light and switch to a Lucifer Yellow filter. Apply a hyperpolarizing current to the dye fill electrode with the amplifier to inject the Lucifer Yellow into the GF. The quantity of dye injected can be controlled by the amount of current passed as well as the duration the current is passed. To prevent the dye from spilling out of the injection site or damage the GF, it is recommended to inject using a low current (approx 3-5 nA) over 1-5 minutes.</li>
 <li>Apply current until the GF terminal in the VNC can be seen clearly (Figure 4A). For trans-synaptic staining of postsynaptic neurons, continue to pass current on until dye can also be seen in postsynaptic neurons. Switch off the current and remove the dye fill electrode.</li>
</ol>
3. Representative Results:
A representative dye fill for one GF with Lucifer Yellow is shown in Figure 4A. The image was acquired as an image stack in 
Nikon Elements software using a spot camera mounted on the microscope. In this sample current was passed through the electrode for about 2 minutes. 
The GF is thoroughly filled and the large GF terminal in the thorax is well labeled (Figure 4A, ellipse). No postsynaptic neurons are visible in this case, 
although Lucifer Yellow is small enough to pass through gap junctions. If the dye is not injected long enough into the GF then trans-synaptic fills of the 
postsynaptic neurons may not been seen because the signal is too weak to be detected. Neurons postsynaptic to the GFs (Figure 4B, arrow heads) can be 
visualized more reliably using even smaller neuronal tracers, such as Neurobiotin. Injecting a 7% Neurobiotin in 2 M potassium acetate solution with a 
depolarizing current for 2 minutes is sufficient to label postsynaptic neurons strongly. Neurobiotin can be visualized for confocal microscopy using 
Streptavidin coupled to dyes of different wavelengths. Because Neurobiotin is not a fluorescent dye, co-injection of Rhodamin-Dextran or Alexa 
hydrazides in the same solution, can be used to monitor a successful injection into the GF (Figure 4C, from Boerner and Godenschwege, 20105).
<img src="/files/ftp_upload/3080/3080fig1.jpg" alt="Figure 1" /> Figure 1. Schematic drawing of a fly viewed laterally. The thorax is displayed with a longitudinal incision to visualize the internal structures. During the dissection, the dorsal longitudinal flight muscles (DLMs, blue) were cut without damaging the ventral nerve cord (VNC, deep red), which lies ventral to the flight muscles. The gut (green) lies above the VNC.
<img src="/files/ftp_upload/3080/3080fig2.jpg" alt="Figure 2" /> Figure 2. Dorsal view of a dissected fly. Two pins are placed behind the head to position the fly. The pins affixed through the flight muscles (DLM) hold the thorax open. Internal organs were removed to expose the ventral nerve cord (VNC).
<img src="/files/ftp_upload/3080/3080fig3.jpg" alt="Figure 3" /> Figure 3. Dye fill setup.
<img src="/files/ftp_upload/3080/3080fig4.jpg" alt="Figure 4" /> Figure 4. A) Z-projection of the posterior part of the VNC showing one GF filled with Lucifer Yellow. The image was acquired immediately after dye filling with a spot camera. In wild-type animals the terminal (ellipse) in the thorax is clearly visible. B) Confocal image of a GF dye fill with Neurobiotin in a wild-type fly. Both GFs (arrows) and neurons postsynaptic to the GFs (arrow heads) are labeled. Neurobiotin was visualized with Streptavidin coupled to Cy3. C) Confocal image of wild-type GFs filled with Alexa555.

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

No conflicts of interest declared.

The nervous system can be dissected without extracting it from the fly body. This has two advantages, first, the dissection causes little damage to the nervous system, and second, most nerves stay attached to the muscles and sensory organs. Performing the dissection, as described, prepares the sample for straight forward dye-labeling of the GFs. Conveniently, the motoneurons postsynaptic to the GFs stay attached to the muscles. Axons, therefore, are undamaged, keeping the neurons alive longer. In addition, damage to the ...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>AmScope</td>
			<td>SM-2TZ</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vannas Scissors Superfine</td>
			<td>Academic Instruments</td>
			<td>VS1023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont Forceps Dumontstar 55</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Austerlitz Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>&Oslash; 0.1mm</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Electrodes</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vertical Pipette Puller 700c</td>
			<td>David Kopf Instruments</td>
			<td>Model 700C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucifer Yellow CH dilithium salt</td>
			<td>Sigma</td>
			<td>L0259</td>
			<td>1% in H2O</td>
		</tr>
		<tr>
			<td>Fixed Stage Upright Microscope & Camera</td>
			<td>Nikon</td>
			<td>FN-1 & DS-U2 Camera</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plan Fluor 10X Air Objective</td>
			<td>Nikon</td>
			<td>CFI Plan Fluor 10X NA 0.3 WD 16mm</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluor 40X water dipping Objective</td>
			<td>Nikon</td>
			<td>CFI Fluor 40X W NA 0.80 WD 2.0mm</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3D hydraulic Micromanipulator</td>
			<td>Narishige International</td>
			<td>Model MW0-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Getting Instruments, Inc.</td>
			<td>Model 5A</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

whole-mount-preparation-adult-drosophila-ventral-nerve-cord-for-giant

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18.9K Views.  Florida Atlantic University. An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Video: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...